Frédéric Darriet

Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s. s.

Pyrethroid‐impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage‐gated sodium channel protein recently shown to be associated with mutations of the para‐type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR‐based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

Combined pyrethroid and carbamate 'two-in-one' treated mosquito nets : field efficacy against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus

A new approach is proposed in the treatment of mosquito nets, using a ‘two‐in‐one’ combination of pyrethroid and non‐pyrethroid insecticides applied to different parts of bednets. The objectives are mainly to overcome certain limitations of pyrethroid‐impregnated bednets currently recommended for malaria control purposes. Apart from developing alternatives to pyrethroid dependency, we sought to counteract pyrethroid irritant effects on mosquitoes (excito‐repellency) and resistance to pyrethroids. The idea takes advantage of the presumed host‐seeking behaviour of mosquitoes confronted by a net draped over a bed, whereby the mosquito may explore the net from the top downwards. Thus, nets could be more effective if treated on the upper part with residual non‐irritant insecticide (carbamate or organophosphate) and with a pyrethroid on the lower part. Sequential exposure to different insecticides with distinct modes of action is equivalent to the use of a mixture as a potential method of managing insecticide resistance. We also intended to improve the control of nuisance mosquitoes, especially Culex quinquefasciatus Say (Diptera: Culicidae) that often survive pyrethroids, in order to encourage public compliance with use of insecticide‐treated nets (ITNs).

Insecticide Resistance in the Dengue Vector Aedes aegypti from Martinique: Distribution, Mechanisms and Relations with Environmental Factors

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the “knock-down resistance” V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

Efficacy of mosquito nets treated with insecticide mixtures or mosaics against insecticide resistant Anopheles gambiae and Culex quinquefasciatus (Diptera: Culicidae) in Côte d'Ivoire.

Only pyrethroid insecticides have so far been recommended for the treatment of mosquito nets for malaria control. Increasing resistance of malaria vectors to pyrethroids threatens to reduce the potency of this important method of vector control. Among the strategies proposed for resistance management is to use a pyrethroid and a non-pyrethroid insecticide in combination on the same mosquito net, either separately or as a mixture. Mixtures are particularly promising if there is potentiation between the two insecticides as this would make it possible to lower the dosage of each, as has been demonstrated under laboratory conditions for a mixture of bifenthrin (pyrethroid) and carbosulfan (carbamate). The effect of these types of treatment were compared in experimental huts on wild populations of Anopheles gambiae Giles and the nuisance mosquito Culex quinquefasciatus Say, both of which are multi-resistant. Four treatments were evaluated in experimental huts over six months: the recommended dosage of 50 mg m(-2) bifenthrin, 300 mg m(-2) carbosulfan, a mosaic of 300 mg m(-2) carbosulfan on the ceiling and 50 mg m(-2) bifenthrin on the sides, and a mixture of 6.25 mg m(-2) carbosulfan and 25 mg m(-2) bifenthrin. The mixture and mosaic treatments did not differ significantly in effectiveness from carbosulfan and bifenthrin alone against anophelines in terms of deterrency, induced exophily, blood feeding inhibition and overall mortality, but were more effective than in earlier tests with deltamethrin. These results are considered encouraging, as the combination of different classes of insecticides might be a potential tool for resistance management. The mixture might have an advantage in terms of lower cost and toxicity.

Lethal and behavioural effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti mosquitoes in laboratory assays

Abstract The knock‐down, mortality and ‘irritancy’ effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti (L) (Diptera: Culicidae) were evaluated in the laboratory in the absence of animal bait. Filter paper tests were carried out to assess the knock‐down effect (KDt50 and KDt95) and mortality (LC50 and LC95) induced by each repellent. ‘Irritancy’ tests were carried out to compare the flight response (time to first take‐off, or FT) to increasing concentrations of repellents (2–7%) and at five distances from the treated surface (0–40 mm). DEET had an insecticidal effect (KDt50= 9.7 min at 7%; CL50= 1165 mg/m2), whereas IR3535 and KBR 3023 did not. Relative to an untreated control, IR3535 was an irritant (relative irritancy or RI > 1) at doses of 5% and 7% (RI = 17.7 and 9.9, respectively), whereas DEET was an irritant at lower concentrations (RI = 12.3 at 2% DEET). KBR 3023 was the weakest irritant over the same range of concentrations (RImax= 3.6 at 6%). DEET was more of an irritant (RI20= 9.4) than IR3535 (RI20= 2.9) over a range of distances (0–20 mm), and KBR 3023 was not an irritant unless mosquitoes made contact with the treated surface. All three repellents had a significant effect on mosquitoes, but DEET exhibited a more complex mode of action than the others due to its insecticidal properties. The repellents do not behave as a single class of compounds with a common mode of action, but most probably affect different physiological systems in insects. The physiological and molecular mechanisms of repellents, especially DEET, should be investigated to ensure a better use of these molecules for skin applications and/or for treating materials against mosquitoes.

Comparative performances, under laboratory conditions, of seven pyrethroid insecticides used for impregnation of mosquito nets

OBJECTIVE To compare the efficacy of seven pyrethroid insecticides for impregnation of mosquito nets, six currently recommended by WHO and one candidate (bifenthrin), under laboratory conditions. METHODS Tests were conducted using pyrethroid-susceptible and pyrethroid-resistant strains of Anopheles gambiae and Culex quinquefasciatus. Knock-down effect, irritancy and mortality were measured using standard WHO cone tests. Mortality and blood-feeding inhibition were also measured using a baited tunnel device. FINDINGS For susceptible A. gambiae, alpha-cypermethrin had the fastest knock-down effect. For resistant A. gambiae, the knock- down effect was slightly slower with alpha-cypermethrin and much reduced following exposure to the other insecticides, particularly bifenthrin and permethrin. For susceptible C. quinquefasciatus, the knock-down effect was significantly slower than in A. gambiae, particularly with bifenthrin, and no knock-down effect was observed with any of the pyrethroids against the resistant strain. Bifenthrin was significantly less irritant than the other pyrethroids to susceptible and resistant A. gambiae but there was no clear ranking of pyrethroid irritancy against C. quinquefasciatus. In tunnels, all insecticides were less toxic against C. quinquefasciatus than against A. gambiae for susceptible strains. For resistant strains, mortality was significant with all the pyrethroids with A. gambiae but not with C. quinquefasciatus. Inhibition of blood-feeding was also high for susceptible strains of both species and for resistant A. gambiae but lower for resistant C. quinquefasciatus; bifenthrin had the greatest impact. CONCLUSIONS Efficacy for impregnation of mosquito nets against A. gambiae was greatest with alpha-cypermethrin. Bifenthrin is likely to have a significant comparative advantage over other pyrethroids in areas with pyrethroid resistance because of its much stronger impact on the nuisance mosquito, C. quinquefasciatus, despite its slower knock-down effect and irritancy. Selection of pyrethroids for mosquito vector control and personal protection should take into account the different effects of these insecticides, the status of pyrethroid resistance in the target area, and the importance of nuisance mosquitoes, such as C. quinquefasciatus.

Laboratory Evaluation of Pyriproxyfen and Spinosad, Alone and in Combination, Against Aedes aegypti Larvae

Abstract In this study, the efficacy of pyriproxyfen and spinosad, alone and in combination, was evaluated against the dengue vector Aedes aegypti (L.). Larval bioassays were carried out on susceptible mosquito larvae to determine the concentration–mortality responses of mosquitoes exposed to each insecticide alone and in mixture. Synergism between pyriproxyfen and spinosad was determined by the calculation of a combination index (CI) by using the isobologram method. For pyriproxyfen, LC50 and LC95 were 1.1 × 10−4 (1.0 × 10−4–1.1 × 10−4) and 3.2 × 10−4 (2.9 × 10−4–3.6 × 10−4) mg/liter, respectively. Pyriproxyfen acted at very low concentrations by inhibiting the adult emergence of Ae. aegypti (97% inhibition rates at 3.3 × 10−4 mg/liter). Spinosad activity was ≈500 times lower than that of pyriproxyfen against the Bora strain, with LC50 and LC95 values estimated at 0.055 (0.047–0.064) and 0.20 (0.15–0.27) mg/liter, respectively. A binary mixture of pyriproxyfen and spinosad was realized at the ratio 1:500 by considering the values of the LC50 obtained for each product. The LC50 and LC95 of the mixture were 0.019 (0.016–0.022) and 0.050 (0.040–0.065) mg/liter, respectively. The mixture combined both the larvicidal activity of spinosad and the juvenoid action of pyriproxyfen. From the LC70 to LC99, a significant synergism effect was observed between the two insecticides (CI ranged from 0.74 to 0.31). This strong synergism observed at high concentrations allows a reduction by five and nine-fold of pyriproxyfen and spinosad amounts to kill almost 100% mosquitoes. Combination of pyriproxyfen and spinosad may then represent a promising strategy to improve mosquito control in situations with insecticide-resistant Aedes dengue vectors.

Field evaluation of pyriproxyfen and spinosad mixture for the control of insecticide resistant Aedes aegypti in Martinique (French West Indies)

BackgroundThe resistance of Ae. aegypti to insecticides is already widespread and continues to develop. It represents a serious problem for programmes aimed at the control and prevention of dengue in tropical countries. In the light of this problem measures to control Ae. aegypti are being orientated towards how best to use existing insecticides, notably by combining those that have different modes of action.ResultsIn this study we evaluated the operational efficiency of a mixture composed of pyriproxyfen (an insect growth regulator) and spinosad (a biopesticide) against a population of Ae. aegypti from Martinique resistant to pyrethroid and organophosphate insecticides. The first step consisted of evaluating the efficacy of pyriproxyfen and spinosad when used alone, or in combination, against Ae. aegypti larvae under simulated conditions. The results showed that the mixture of pyriproxyfen+spinosad remained active for at least 8 months, compared with 3 months for spinosad alone, and 5 months for pyriproxyfen alone. In a second step in containers experiencing natural conditions, pyriproxyfen and spinosad, maintained the rate of adult emergence at 20% for 3 weeks and 3.5 months, respectively. Following the same criteria of evaluation, the mixture pyriproxyfen+spinosad remained effective for 4.5 months, showing that the combination of the two larvicides with different modes of action acted to increase the residual activity of the treatment.ConclusionThe mixture of pyriproxyfen and spinosad kills larvae and pupae giving it a broader range of action than either insecticide. This mixture could preserve the utility of both insecticides in public health programs.

Spinosad: a new larvicide against insecticide-resistant mosquito larvae.

ABSTRACT Among the characteristics of spinosad that are worth noting is its environmentally favorable toxicity profile. It is a compound of biological origin, whose insecticide action is highly effective against a great number of insects. Laboratory larval bioassays of spinosad on Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae (specimens that were either susceptible or resistant to pyrethroids, carbamates, and organ-ophosphates) showed that this product had a lethal action (mortality after 24 h of exposure) regardless of the original status, susceptible or resistant, of the mosquito larvae.

Field Efficacy of New Larvicide Products for Control of Multi-Resistant Aedes aegypti Populations in Martinique (French West Indies)

World-wide dengue vector control is hampered by the spread of insecticide resistance in Aedes aegypti. We report the resistance status of a wild Ae. aegypti population from Martinique (Vauclin) to conventional larvicides (Bacillus thuringiensis var israeliensis [Bti] and temephos) and potential alternatives (spinosad, diflubenzuron, and pyriproxyfen). The efficacy and residual activity of these insecticides were evaluated under simulated and field conditions. The Vauclin strain exhibited a high level of resistance to temephos, a tolerance to insect growth regulators, and full susceptibility to spinosad and Bti. In simulated trials, pyriproxyfen and Bti showed long residual activities in permanent breeding containers (28 and 37 weeks), whereas under field conditions they failed to curtail Ae. aegypti populations after four weeks. Conversely, diflubenzuron and spinosad showed a residual efficacy of 16 weeks, suggesting that these chemicals may be promising alternatives to Bti and temephos for controlling insecticide-resistant Ae. aegypti populations.